Anticancer composition comprising tumor-specific oncolytic adenovirus and immune checkpoint inhibitor

ABSTRACT

The present invention relates to an anticancer composition comprising a tumor-specific oncolytic adenovirus and an immune checkpoint inhibitor. The recombinant adenovirus having IL-12 and shVEGF, or IL-12 and GM-CSF-RLX inserted therein, according to the present invention, exhibits an excellent anticancer effect by enhancing immune functions, and such anticancer effect has been confirmed to be notably enhanced through concomitant administration with an immune checkpoint inhibitor, and thus the present invention may be used as a key technique in the field of cancer treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Rule 53(b) Continuation of U.S. application Ser.No. 16/553,275, filed on Aug. 28, 2019, which is a Continuation ofInternational Application No. PCT/KR2018/002439, filed on Feb. 28, 2018,which claims priority based on Korean Patent Application No.10-2017-0026339, filed on Feb. 28, 2017, the respective disclosures ofwhich are incorporated herein by reference in their entireties.

TECHNICAL FIELD

The present invention relates to a tumor-specific oncolytic adenoviruswhich expresses cytokines and degradation factors for the extracellularmatrix, an anticancer composition comprising the tumor-specificoncolytic adenovirus and an immune checkpoint inhibitor.

BACKGROUND

Despite the rapid development of cancer therapeutic agents, cancer isstill one of the diseases with a high death rate worldwide. The maincancer treatment methods conventionally used clinically are surgery,radiation treatment, and anticancer agent treatment methods, or methodsof maximally removing cancer cells from patients as a treatment inparallel with the same. However, these treatments exhibit treatmenteffects only when cancer cells are completely removed in a state where arelatively early cancer is not metastasized, and also kill other normalcells which are rapidly dividing, and thus have a disadvantage in thatvarious side effects may be caused. Accordingly, studies have beenrecently conducted on the treatment of cancer using the tumor-specificimmune activity of the body as an immunotherapeutic method.

However, it is difficult to perform the immune treatment because cancerhas the ability not only to cleverly evade and destroy various hostimmune responses, and as a result continuously maintains the survival oftumors by inducing the immunosuppressive tumor microenvironment, butalso to escape from the activated anti-tumor immune response even thoughthe immune system is activated. Accordingly, in order to enhance theimmune response against cancer cells by improving the immunosuppressivetumor microenvironment, studies using cytokine genes such as IL-12,IL-18, IL-23, interferon-gamma (IFN-γ), a granulocyte-macrophagecolony-stimulating factor (GM-CSF), a tumor necrosis factor-alpha(TNF-α), costimulatory factors such as B7 molecules, dendritic cells(DCs) directly serving as antigen presenting cells (APCs), T cellsactivated by tumor antigens, natural killer cells (NKCs), and the likehave been carried out in various directions. It is known that IL-12 isproduced by APCs such as monocytes, macrophages, and DCs, and bydirectly acting on cytotoxic T-lymphocytes (CTLs) and NK cells that caneffectively remove cancer cells activates them, stimulates production ofIFN-γ, as well as enhances an ability to kill cancer cells by directlyacting on them. In addition, IL-12 plays an important role in promotingdifferentiation into T helper 1 (TH1) cells by acting on naïve CD4⁺lymphocytes, and thus, activating an anticancer immune response byinducing and enhancing the cell-mediated immune response which plays apivotal role in the anticancer immune response. Further, VEGF is asignal protein produced by cells that promote angiogenesis andvascularization, and affects T cell precursors in the bone marrow to notonly suppress the proliferation and maturation of T cells and dendriticcells, but also play an important function in vascularization, therebyexhibiting a side effect called cancer metastasis. Accordingly, VEGF isnot only a promoter of tumor growth, but also acts as a suppressor inanticancer immunity, and VEGF downregulation by VEGF shRNA (shorthairpin ribonucleic acid) is expected to restore immune responses and toincrease anticancer effects. Furthermore, GM-CSF serves to strengthenthe immune responses of CD4⁺ and CD8⁺ T cells by stimulating DCs topromote the differentiation of DCs into APCs, and is also involved inthe expression regulation of molecules constituting majorhistocompatibility complex (MHC) class II in primary monocytes. Inaddition, it has been reported that a strong anticancer immune responseis induced by inducing many APCs to gather around tumors due to theeffect of expression of GM-CSF in the tumors to effectively processtumor antigens.

Based on this, our laboratory also reported an anti-tumor effect ofIL-12 using YKL-1 [Ad-E1B55] as an E1B-55 kDa gene-deletedtumor-selective oncolytic adenovirus, and also reported an anti-tumoreffect of GM-CSF using an RdB adenovirus with a tumor-selective killingability more enhanced, due to the deletion of the E1B gene and amodification in an Rb binding site of E1A. However, although theimmunosuppressive tumor microenvironment is improved, there are stillmany limitations in completely treating cancer due to the lowimmunogenicity of tumors using an immunotherapeutic method.

SUMMARY OF THE INVENTION

As a result of intensive research on developing an immunotherapy forovercoming the immune surveillance evasion of tumors, the presentinventors not only confirmed an excellent anticancer effect of IL-12 anda shVEGF (vascular endothelial growth factor (VEGF)-targeting shorthairpin ribonucleic acid)) or IL-12 and GM-CSF-relaxin in vivo bypreparing a recombinant adenovirus simultaneously expressing them, butalso confirmed that an anticancer effect of the recombinant adenoviruswas improved through co-administration with an immune checkpointinhibitor, thereby completing the present invention based on this.

Accordingly, an object of the present invention is to provide arecombinant adenovirus simultaneously expressing IL-12 and shVEGF, orIL-12 and GM-CSF-Relaxin.

Another object of the present invention is to provide a medicinal use ofthe recombinant adenovirus for the prevention or treatment of cancer.

Still another object of the present invention is to provide a medicinaluse of the recombinant adenovirus for the prevention or treatment ofcancer as a combined formulation with an immune checkpoint inhibitor.

However, technical problems to be achieved by the present invention arenot limited to the aforementioned problems, and other problems that arenot mentioned may be clearly understood by those skilled in the art fromthe following description.

In order to achieve the objects of the present invention as describedabove, the present invention provides a recombinant adenoviruscomprising a gene encoding interleukin 12 (IL-12); a gene encoding agranulocyte-macrophage colony-stimulating factor (GM-CSF); and a geneencoding relaxin, or a recombinant adenovirus comprising a gene encodingInterleukin 12 (IL-12); and a gene encoding a shRNA suppressing theexpression of VEGF.

As an embodiment of the present invention, the recombinant adenovirusmay have one or more regions selected from the group consisting of theE1 and E3 regions deleted.

As another embodiment of the present invention, the gene encoding IL-12may comprise an IL-12A (p35) gene sequence, an IRES sequence, and anIL-12B (p40) gene sequence, and may be inserted into the E1 or E3 regionof the recombinant adenovirus.

As another embodiment of the present invention, the gene encoding IL-12may comprise an IL-12A (p35) gene sequence, inker, and an IL-12B (p40)gene sequence, and may be inserted into the E1 or E3 region of therecombinant adenovirus. As still another embodiment of the presentinvention, the genes encoding GM-CSF and relaxin may comprise a GM-CSFgene sequence, an IRES sequence, and a relaxin gene sequence, and may beinserted into the E1 or E3 region of the recombinant adenovirus.

As yet another embodiment of the present invention, the gene encoding ashRNA suppressing the expression of VEGF may bind complementarily tomRNA of VEGF, and may be inserted into the E1 or E3 region of therecombinant adenovirus.

Further, the present invention provides a pharmaceutical composition forpreventing or treating cancer, the composition including: therecombinant adenovirus; and a pharmaceutically acceptable carrier.

As an embodiment of the present invention, the composition may beco-administered with any one immune checkpoint inhibitor selected fromthe group consisting of a PD-1 (programmed cell death protein 1)antagonist, a PD-L1 (programmed cell death protein ligand 1) antagonist,a PD-L2 antagonist, a CD27 (cluster of differentiation 27) antagonist, aCD28 antagonist, a CD70 antagonist, a CD80 antagonist, a CD86antagonist, a CD137 antagonist, a CD276 antagonist, a KIRs (killer-cellimmunoglobulin-like receptors) antagonist, a LAG3 (lymphocyte-activationgene 3) antagonist, a TNFRSF4 (tumor necrosis factor receptorsuperfamily, member 4) antagonist, a GITR (glucocorticoid-inducedTNFR-related protein) antagonist, a GITRL (GITR ligand) antagonist, a4-1BBL (4-1BB ligand) antagonist, a CTLA-4 (cytotoxic T lymphocyteassociated antigen 4) antagonist, an A2AR (adenosine A2A receptor)antagonist, a VTCN1 (V-set domain-containing T-cell activationinhibitor 1) antagonist, a BTLA (B- and T-lymphocyte attenuator)antagonist, an IDO (Indoleamine 2,3-dioxygenase) antagonist, a TIM-3(T-cell immunoglobulin and mucin-domain containing-3) antagonist, aVISTA (V-domain Ig suppressor of T cell activation) antagonist, a KLRAantagonist, and a combination thereof.

As another embodiment of the present invention, the composition mayenhance anti-tumor immunity.

As still another embodiment of the present invention, the cancer may beselected from the group consisting of gastric cancer, lung cancer,non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer,bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreaticcancer, bladder cancer, colon cancer, cervical cancer, bone cancer,non-small cell bone cancer, hematologic malignancy, skin cancer, head orneck cancer, uterine cancer, colorectal cancer, anal near cancer, coloncancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvacancer, Hodgkin's disease, esophageal cancer, small intestine cancer,endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer,soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer,chronic or acute leukemia, lymphocytic lymphoma, kidney or hydrouretercancer, renal cell carcinoma, renal pelvic carcinoma, salivary glandcancer, sarcoma cancer, pseudomyxoma peritonei, hepatoblastoma,testicular cancer, glioblastoma, cheilocarcinoma, ovarian germ celltumors, basal cell carcinoma, multiple myeloma, gallbladder cancer,choroidal melanoma, cancer of the ampulla of Vater, peritoneal cancer,tongue cancer, small cell cancer, pediatric lymphoma, neuroblastoma,duodenal cancer, ureteral cancer, astrocytoma, meningioma, renal pelviscancer, pudendum cancer, thymus cancer, central nervous system tumors,primary central nervous system lymphoma, spinal cord tumors, brain stemneuroglioma, and pituitary adenoma, and the cancer may also be arecurrent cancer.

The recombinant adenovirus having IL-12 and shVEGF, or IL-12 andGM-CSF-Relaxin inserted therein, according to the present invention,exhibits an excellent anticancer effect by improving immune functions,and such an anticancer effect has been confirmed to be remarkablyenhanced through co-administration with an immune checkpoint inhibitor,and thus the present invention may be used as a key technique in thefield of cancer treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentdisclosure will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the accompanying drawings, in which:

FIG. 1 illustrates characteristics of an oncolytic adenovirussimultaneously expressing IL-12 and shRNA for VEGF, and is a viewschematically illustrating the genetic structure of RdB/IL12/shVEGF Ad.An RdB comprises a modified E1A (mutation of open star-Rb proteinsbinding site), and E1B-19 and E1B-55 kDa (E1B), and E3 region (E3) aredeleted; and a mouse IL-12 and a mouse shVEGF are inserted into the E1or E3 region of the adenoviral genome, respectively.

FIG. 2 illustrates characteristics of an oncolytic adenovirussimultaneously expressing IL-12 and GM-CSF and relaxin (GMCSF-Relaxin),and is a view schematically illustrating the genetic structure ofRdB/IL12/GMCSF-RLX Ad. IL-12 and GMCSF-Relaxin are inserted into the E1or E3 region of the adenoviral genome, respectively. GMCSF and relaxinmay also be expressed by an expression system linked by an IRES(internal ribosome entry site), and in the present invention, the IRESis a regulatory sequence found in the RNAs of several viruses and cells.

FIGS. 3A and 3B confirm an anticancer effect according to theadministration of RdB/IL12/shVEGF Ad and the co-administration ofRdB/IL12/shVEGF Ad and an immune checkpoint inhibitor (anti PD-L1antibody; αPD-L1) in mice with melanoma, FIG. 3A is a result confirminga change in volume of tumors, and FIG. 3B is a result confirming achange in survival rate of mice with melanoma. Ad 5×10⁹ viral particle(VP) (FIG. 3A; an initial tumor volume: 80 to 100 mm³) adenovirus wasadministered to tumors of C57BL/6 mice on day 1, 3, and 5, and 200 μg ofan anti PD-L1 antibody was intraperitoneally administered. The volume oftumors was monitored and recorded daily until the end of the experiment.The red arrow indicates the time when the adenovirus is injected, andthe black arrow indicates the time when the antibody is injected.

FIGS. 4A and 4B confirm an anticancer effect according to theadministration of RdB/IL12/GMCSF-RLX Ad and the co-administration ofRdB/IL12/GMCSF-RLX Ad and an immune checkpoint inhibitor (anti PD-L1antibody) in mice with melanoma, FIG. 4A is a result confirming a changein volume of tumors, and FIG. 4B is a result confirming a change insurvival rate of mice with melanoma.

FIG. 5 confirms an anticancer effect according to the administration ofRdB/IL12/shVEGF Ad and the co-administration of RdB/IL12/shVEGF Ad andan immune checkpoint inhibitor (anti PD-1 antibody) in mice withmelanoma, and is a result confirming a change in volume of tumors whenRdB/IL12/shVEGF Ad is administered at a relatively low dose of 1×10⁹ VP.

FIG. 6 confirms an anticancer effect according to the administration ofRdB/IL12/GMCSF-RLX Ad and the co-administration of RdB/IL12/GMCSF-RLX Adand an immune checkpoint inhibitor (anti PD-1 antibody) in mice withmelanoma, and is a result confirming a change in volume of tumors whenRdB/IL12/GMCSF-RLX Ad is administered at a relatively low dose of 1×10⁹VP.

FIG. 7 is a result confirming an anticancer effect according to theco-administration of HmT-Rd19-K35/IL21 Ad, RdB/GMCSF/shVEGF Ad, andYKL-1/GMCSF/B7.1 Ad and an immune checkpoint inhibitor (anti PD-1antibody) in mice with melanoma.

FIGS. 8A and 8B confirm an anticancer effect according to theadministration of RdB/IL12/GMCSF-RLX Ad and the co-administration ofRdB/IL12/GMCSF-RLX Ad and an immune checkpoint inhibitor (anti PD-1antibody) in mice with melanoma, FIG. 8A is a result confirming a changein volume of tumors, and FIG. 8B is a result confirming a change insurvival rate of mice with melanoma.

FIGS. 9A and 9B confirm an anticancer effect according to theadministration of RdB/IL12/GMCSF-RLX Ad and the co-administration ofRdB/IL12/GMCSF-RLX Ad and an immune checkpoint inhibitor (anti CTLA-4antibody) in mice with melanoma, FIG. 9A is a result confirming a changein volume of tumors, and FIG. 9B is a result confirming a change insurvival rate of mice with melanoma.

FIGS. 10A and 10B confirm an anti-tumor immune memory effect accordingto the administration of RdB/IL12/shVEGF and the co-administration ofRdB/IL12/shVEGF Ad and an immune checkpoint inhibitor (anti PD-L1antibody) in mice with melanoma, FIG. 10A is a result confirming achange in volume of secondary tumors when RdB/IL12/shVEGF Ad isadministered, and FIG. 10B is a result confirming a change in volume ofsecondary tumors when RdB/IL12/shVEGF Ad and an immune checkpointinhibitor (anti PD-1 antibody) are co-administered.

FIG. 11 is a result confirming, through enzyme-linked immunosorbentassay (ELISA), a change in expression of IL-12 and GMCSF according tomultiplicity of infection (MOI) after a Syrian hamster pancreatic cancercell line (Hap-T1) is infected with RdB/IL12/GMCSF-RLX Ad.

FIG. 12 is a result confirming, through reverse transcription polymerasechain reaction (RT-PCR), a change in expression of relaxin according toMOI after a Syrian hamster pancreatic cancer cell line (Hap-T1) isinfected with RdB/IL12/GMCSF-RLX Ad.

FIG. 13 is a result confirming a change in volume of tumors according tothe co-administration of RdB/IL12/GMCSF-RLX Ad and an immune checkpointinhibitor (anti PD-1 antibody) in a Syrian hamster tumor animal model.

FIG. 14 is a result confirming, by an immunohistological method, achange in tumor tissues according to the co-administration ofRdB/IL12/GMCSF-RLX Ad and an immune checkpoint inhibitor (anti PD-1antibody) in a Syrian hamster tumor animal model.

FIG. 15 is a result confirming a change in population of interferon(IFN)-y-expressing CD4⁺ or CD8⁺ T cells according to theco-administration of RdB/IL12/GMCSF-RLX Ad and an immune checkpointinhibitor (anti PD-1 antibody) in a draining lymph node (DLN).

FIG. 16 is a result confirming a change in population of interferon(IFN)-y-expressing CD4⁺ or CD8⁺ T cells according to theco-administration of RdB/IL12/GMCSF-RLX Ad and an immune checkpointinhibitor (anti PD-1 antibody) in lymphocytes infiltrated into tumortissues.

FIG. 17 is a result confirming a change in expression of IFN-γ in tumortissues according to the co-administration of RdB/IL12/GMCSF-RLX Ad andan immune checkpoint inhibitor (anti PD-1 antibody).

FIG. 18 is a result confirming a change in volume of tumors according tothe co-administration of RdB/IL12/GMCSF-RLX Ad and an immune checkpointinhibitor (anti PD-1 antibody) under a condition of resistance to theimmune checkpoint inhibitor.

FIG. 19 is a result confirming a change in volume of tumors on day 23after administration according to the co-administration ofRdB/IL12/GMCSF-RLX Ad and an immune checkpoint inhibitor (anti PD-1antibody) under a condition of resistance to the immune checkpointinhibitor.

FIG. 20 confirms a change in infiltration of an immune checkpointinhibitor (anti PD-1 antibody) in tumor tissues by the administration ofRdB/IL12/GMCSF-RLX Ad, and is a result of quantifying the accumulationof Alexa 488-αPD-1 in tumor tissues through an immunofluorescenceanalysis.

FIG. 21 confirms a change in infiltration of an immune checkpointinhibitor (anti PD-1 antibody) in tumor tissues by the administration ofRdB/IL12/GMCSF-RLX Ad, and is a result confirming whether Alexa488-αPD-1 is located in an expression site of CD4+ or CD8+ T cellsthrough immunofluorescence.

FIG. 22 confirms a change in infiltration of an immune checkpointinhibitor (anti PD-1 antibody) by the administration ofRdB/IL12/GMCSF-RLX Ad on a systemic basis, and is a result of analyzingan immune positron emission tomography (PET) image over time.

FIG. 23 confirms a change in infiltration of an immune checkpointinhibitor (anti PD-1 antibody) by the administration ofRdB/IL12/GMCSF-RLX Ad on a systemic basis, and is a result of evaluatingand comparing the distribution or absorption of ⁶⁴CU-αPD-1 in varioustissues including tumor tissues by % ID/g.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant adenovirus including: agene encoding Interleukin 12 (IL-12); a gene encoding agranulocyte-macrophage colony-stimulating factor (GM-CSF); and a geneencoding relaxin, or a recombinant adenovirus including: a gene encodingIL-12; and a gene encoding a shRNA suppressing the expression of VEGF.

Although an immune gene therapy for cancer has developed as a morepromising approach, tumors have also created numerous strategies toavoid the immune surveillance system. In order to overcome theseobstacles and enhance the effects of anticancer immunity, an oncolyticadenovirus (RdB/IL12/shVEGF Ad, RdB/IL12/GMCSF-RLX Ad) systemsimultaneously expressing IL-12 and a vascular endothelial growth factor(VEGF) short hairpin RNA (shRNA) or IL-12 and GM-CSF-Relaxin wasconstructed as an appropriate therapeutic adjuvant for restoringimmunosuppression and increasing anticancer immunity. IL-12 serves toincrease anti-tumor immunity by inducing the differentiation of T helper1 cells and activating the cytotoxicity of cytotoxic T lymphocytes andnatural killer cells. It is known that shVEGF and GM-CSF serve tostrengthen the immune response of CD4⁺ and CD8⁺ T cells by promoting theproliferation and maturation of T cells and dendritic cells andstimulating DCs to promote the differentiation of DCs into APCs,respectively, and these effects are expected to further amplify theIL-12 function. Thus, the present inventors attempted immune genetherapy using a technical feature of an adenovirus simultaneouslyexpressing IL-2 and shVEGF, or IL-12 and GM-CSF-Relaxin in tumors forthe first time. In an example of the present invention, as a result ofadministering the recombinant adenovirus (RdB/IL12/shVEGF Ad,RdB/IL12/GMCSF-RLX Ad) to a melanoma animal model, it was experimentallyconfirmed that the tumor growth inhibition rate, complete remissionrate, and survival rate of mice were increased.

In addition, IL-12 has drawn much attention as a core cytokine foranticancer immunotherapy, and in particular, in an anticancerimmunotherapeutic method for patients with impaired immunity in thebody, repeated administration of IL-12 has been frequently considered inorder to obtain effective therapeutic efficacy. However, the IL-12 isclinically known to have the cytokine-associated toxicity in the wholebody, particularly, the kidneys, and thus has a technical limitationthat the IL-12 has to be used as a cancer therapeutic agent within alimited dose range. Based on this technical background, the recombinantadenovirus simultaneously expressing the IL-12 gene according to thepresent invention and an additional gene has another technical featureof solving the toxicity problem for normal cells due to the high doseand repeated administration of IL-12, which has been pointed out as alimitation of the anticancer immunotherapy in the related art, byinducing a high oncolytic effect even at a low viral titer.

Accordingly, the recombinant adenovirus of the present invention ischaracterized by including a gene encoding IL-12 and including any oneof a gene encoding GM-CSF, a gene encoding relaxin, and a gene encodinga shRNA suppressing the expression of VEGF.

Interleukin-12 (IL-12) is a disulfide-linked heterodimer which iscomposed of a 40 kDa subunit (p40 subunit, p40) encoded by IL-12B (orInterleukin-12 subunit beta) and a 35 kDa subunit (p35 subunit or p35)encoded by IL-12A (or Interleukin-12 subunit alpha) and has a molecularweight of 75 kDa. IL-12 binds to receptors on the cell surface ofactivated T cells, B cells, and NK cells produced by antigen presentingcells such as macrophages (Desai, B. B., et al., J. Immunol.,148:3125-3132 (1992); Vogel, L. A., et al., Int. Immunol., 8:1955-1962,(1996)). IL-12 has been reported to be an important co-stimulator of Th1clone proliferation (Kennedy et al., Eur. J. Immunol. 24:2271-2278,1994), and is known to increase the production of IgG2a antibodies inserum (Morris, S. C., et al., J. Immunol. 152:1047-1056 (1994); Germann,T. M., et al., Eur. J. Immunol., 25:823-829 (1995); Sher, A., et al.,Ann. N.Y. Acad. Sci., 795:202-207 (1996); Buchanan, J. M., et al., Int.Immunol., 7:1519-1528 (1995); Metzger, D. W. et al., Eur. J. Immunol.,27:1958-1965 (1997)). Further, it has been reported that administrationof IL-12 can temporarily reduce the production of IgG1 antibody,suggesting that IL-12 inhibits the Th2 immune response (Morris, S. C.,et al., J. Immunol. 152:1047-1056 (1994); McKnight, A. J., J. Immunol.152:2172-2179 (1994); Buchanan, J. M., et al., Int. Immunol.,7:1519-1528 (1995)).

Cloning and purification of IL-12 is disclosed in WO 92/05256, WO90/05147, and EP 322,827, the contents of which are incorporated hereinby reference. The recombinant adenoviruses of the present inventioncomprise an IL-12 coding nucleotide sequence in an expressible form andcan secrete IL-12 in tumor cells to enhance antitumor immune responses.

In order to efficiently express IL-12 using the recombinant adenovirusof the present invention, an expression system can be constructed usingboth the p35 subunit coding nucleotide sequence and the p40 subunitcoding nucleotide sequence. In the present invention, the p35 subunitand the p40 subunit comprise not only the subunits illustrated in theembodiments of the present invention but also all the analogues of thesubunits that can perform the unique function of each subunit.

In one embodiment of the present invention, the amino acid sequences ofthe human p35 and p40 subunits that may be used in the present inventionare those sequences of GenBank accession numbers AAD56385 and AAD56386,respectively (if the amino acid sequences of the mouse p35 and p40subunits are to be expressed, AAA39292 and AAA39296).

In one embodiment of the present invention, the nucleotide sequencesencoding the p35 and p40 subunits that may be used in the presentinvention are IL-12A (p35) gene sequence and an IL-12B (p40) genesequence encoding the amino acid sequences of the p35 and p40 subunits.The nucleotide sequences encoding the human p35 and p40 subunits can befound in GenBank accession numbers AF180562 and AF180563, and preferablythe nucleotide sequences corresponding to the coding sequence (CDS) canbe used (If using the mouse p35 and p40 nucleotide sequences, refer tothe CDS sequence in each of the sequences listed in GenBank AccessionNumbers M86672 and M86671).

Also, the “gene encoding IL-12” used in the present invention comprisesan IL-12A (p35) gene sequence and an IL-12B (p40) gene sequence, and maycomprise an IRES sequence or linker sequence between the IL-12A (p35)gene sequence and the IL-12B (p40) gene sequence for the effectivetranslation of a viral protein. Preferably, in one embodiment of thepresent invention, the IL-12A (p35) gene sequence may comprise orconsist of a sequence of SEQ ID No. 1 (mouse), SEQ ID No. 10 (human), orSEQ ID No. 11 (human); the IL-12B (p40) gene sequence may comprise orconsist of a sequence of SEQ ID No. 2 (mouse), SEQ ID No. 12 (human), orSEQ ID No. 13 (human); the IRES sequence may comprise or consist of asequence of SEQ ID No. 3 or SEQ ID No. 15; and the linker sequence maycomprise or consist of a sequence of SEQ ID No. 14.

The gene encoding IL-12 of the present invention may comprise or consista sequence of SEQ ID No. 16 (human IL-12A-linker-IL-12B) or SEQ ID No.17 (human IL-12A-IRES-IL-12B), but are not limited thereto. The IL-12serves to increase anti-tumor immunity by inducing the differentiationof T helper 1 cells and activating the cytotoxicity of cytotoxic Tlymphocytes and natural killer cells.

The “gene encoding GM-CSF” and the “gene encoding relaxin” used in thepresent invention comprise a GM-CSF gene sequence and a relaxin genesequence, respectively, and may also comprise an IRES sequence betweenthe GM-CSF gene sequence and the relaxin gene sequence. Preferably, theGM-CSF gene sequence may comprise or consist of the sequence of SEQ IDNo. 4 (mouse) or SEQ ID No. 18 (human); the relaxin gene sequence maycomprise or consist of the sequence of SEQ ID No. 5 (human); the IRESsequence may comprise or consist a sequence of SEQ ID No. 3 or SEQ IDNo. 15 but are not limited thereto.

The “gene encoding a shRNA suppressing the expression of VEGF” used inthe present invention has a hairpin structure, and refers to a genecapable of mediating RNA interference or gene silencing. The genecomprises a sequence complementary to VEGF mRNA, and the term“complementary” means including not only the 100% complementary case,but also the incomplete complementarity sufficient to be able tosuppress the expression of the VEGF gene through the RNA interferencemechanism, and means preferably 90% complementarity, more preferably 98%complementarity, and most preferably 100% complementarity. In thepresent specification, the case of expressing the 100% complementarityis specifically described as completely complementary. In the presentinvention, although the gene is also described as shVEGF, but is notlimited thereto, the gene may bind complementarily to the mRNA of VEGF,which is represented by SEQ ID No. 6 (mouse) or 7 (human), to suppressthe expression of VEGF. In one embodiment of the present invention, ashRNA sequence suppressing the expression of mouse VEGF may comprise orconsist of SEQ ID No.8 and SEQ ID No.9.

It is interpreted that the gene sequences also comprise a gene sequenceexhibiting substantial identity or substantial similarity. Theaforementioned substantial identity refers to a random sequencediffering from the sequence of the present invention and having at least80% homology, more preferably 90% homology, and most preferably 95%homology to the sequence of the present invention, when it is aligned tocorrespond to the sequence of the present invention as much as possible,and the aligned sequences are analyzed using an algorithm typically usedin the art. The aforementioned substantial similarity collectivelyrefers to all of the changes in a gene sequence, such as deletion orinsertion of one or more bases, which do not affect the object of thepresent invention of minimizing homologous recombination with arecombinant adenovirus vector. Therefore, the gene sequence of thepresent invention is not limited to the sequences of exemplified SEQ IDNOS. 1 to 18, and is interpreted to be included in the scope of therights of the present invention as long as the sequence does notsubstantially affect the activity of the final product desired in thepresent invention.

Meanwhile, as the recombinant adenovirus of the present invention, anoncolytic adenovirus widely known in the art may be used. In an exampleof the present invention, the recombinant adenovirus comprises anactivated E1A gene and an inactivated E1B-19 gene, an inactivated E1B-55gene, or an inactivated E1B-19/E1B-55 gene. In the presentspecification, the term inactivation used in conjunction with a genemeans that the transcription and/or translation of the gene is notnormally performed, and the function of a normal protein encoded by thegene is not exhibited. For example, the inactivated E1B-19 gene is agene incapable of producing the activated E1B-19 kDa protein by amodification (substitution, addition, and partial or whole deletion) inthe gene. The deletion of E1B-19 may increase the ability to kill cells,and the deletion of the E1B-55 gene makes a recombinant adenovirustumor-specific (see Korean Patent Application No. 2002-23760). The term“deletion” used in conjunction with a viral genomic sequence in thepresent specification has a meaning including complete deletion andpartial deletion of the corresponding sequence.

According to an embodiment of the present invention, the recombinantadenovirus comprises an E1A region, has an E1B region, that is, E1B-19kDa and 55 kDa (E1B) deleted, and an E3 region (E3) deleted. Therecombinant adenovirus including the E1A gene will have replicablecharacteristics. The gene encoding IL-12 is inserted into the deletedE1B region of the recombinant adenovirus, and the genes encoding GM-CSFand relaxin or the gene encoding a shRNA suppressing the expression ofVEGF is inserted into the E3 region. Meanwhile, the E1A site has amodification in which the Glu 45 residue is substituted with Gly and amodification and in which the sequence of amino acids 121 to 127 iswholly substituted with Gly, in a nucleotide sequence encoding Rbbinding site located in the E1A gene sequence.

Meanwhile, viruses other than the adenovirus may also be used in thepresent invention. The virus which may be used in the present inventionmay preferably be vaccinia viruses (Puhlmann M. et al., Human GeneTherapy 10:649-657(1999)), lentiviruses (Wang G. et al., J. Clin.Invest. 104(11): R55-62(1999)), or herpes simplex viruses (Chamber R.,et al., Proc. Natl. Acad. Sci USA 92:1411-1415(1995)) but is not limitedthereto.

The recombinant adenovirus used in the present invention comprises apromoter that is operable in animal cells, preferably mammal cells. Apromoter suitable for the present invention comprises a promoter derivedfrom a mammalian virus and a promoter derived from the genome ofmammalian cells, and comprises, for example, a cytomegalovirus (CMV)promoter, a U6 promoter and an H1 promoter, a murine leukemia virus(MLV) long terminal repeat (LTR) promoter, an adenovirus early promoter,an adenovirus late promoter, a vaccina virus 7.5K promoter, an simianvirus 40 (SV40) promoter, a Herpes simplex virus-thymidine kinase (HSVtk) promoter, an rous sarcoma virus (RSV) promoter, an elongationfactor-1 alpha (EF1 α) promoter, a methallothionin promoter, a β-actinpromoter, a human IL-2 gene promoter, a human IFN gene promoter, a humanIL-4 gene promoter, a human lymphotoxin gene promoter, a human GM-CSFgene promoter, an inducible promoter, a cancer cell-specific promoter[for example, a telomerase reverse transcriptase (TERT) promoter, amodified TERT promoter, a prostate-specific antigen (PSA) promoter, aprostate-specific membrane antigen (PSMA) promoter, a carcinoembryonicantigen (CEA) promoter, a Survivin promoter, an E2F promoter, a modifiedE2F promoter, an alpha-fetoprotein (AFP) promoter, a modified AFPpromoter, an E2F-AFP hybrid promoter, and an E2F-TERT hybrid promoter],a tissue-specific promoter (for example, an albumin promoter), a humanphosphoglycerate kinase (PGK) promoter, and a mouse phosphoglyceratekinase (PGK) promoter, but is not limited thereto. Most preferably, thepromoter is a CMV promoter. It is preferred that in an expressionconstruct for expressing a trans gene, a polyadenylation sequence bindsdownstream of the trans gene. The polyadenylation sequence comprises abovine growth hormone terminator (Gimmi, E. R., et al., Nucleic AcidsRes. 17:6983-6998(1989)), an SV40-derived polyadenylation sequence(Schek, N, et al., Mol. Cell Biol. 12:5386-5393(1992)), HIV-1 polyA(Klasens, B. I. F., et al., Nucleic Acids Res. 26:1870-1876(1998)),3-globin polyA (Gil, A., et al, Cell 49:399-406(1987)), HSV TK polyA(Cole, C. N. and T. P. Stacy, Mol. Cell. Biol. 5:2104-2113(1985)), orpolyomavirus polyA (Batt, D. B and G. G. Biol. 15:4783-4790(1995)), butis not limited thereto.

In the recombinant adenovirus used in the present invention, the IL-12gene sequence, the GM-CSF-Relaxin gene sequence, or the shVEGF genesequence are operably linked to a promoter. As used herein, the term“operably linked” refers to a functional binding between a nucleic acidexpression regulatory sequence (for example: an array of a promoter, asignal sequence, and a transcription regulating factor-binding site) anda different nucleic acid sequence, and accordingly, the regulatorysequence regulates the transcription and/or translation of the differentnucleic acid sequence.

The recombinant adenovirus of the present invention may further comprisean antibiotic resistance gene and a reporter gene (for example, greenfluorescence protein (GFP), luciferase and 3-glucuronidase) as selectivemarkers. The antibiotic resistance gene comprises an antibioticresistance gene typically used in the art, for example, a gene impartingresistance to ampicillin, gentamycin, carbenicillin, chloramphenicol,streptomycin, kanamycin, geneticin, neomycin and tetracycline, andpreferably, is a neomycin resistance gene. The aforementioned selectivemarker may be expressed even in an expression system connected by aseparate promoter or an IRES (internal ribosome entry site), 2A systems(F2A system, P2A system, T2A system), and the IRES that may be used inthe present invention is a regulatory sequence that is found in RNAs ofsome types of viruses and cells.

Another aspect of the present invention provides: a pharmaceuticalcomposition for preventing or treating cancer, the composition includingthe recombinant adenovirus; and a pharmaceutically acceptable carrier; amedicinal use of the recombinant adenovirus for the prevention ortreatment of cancer; and a method for treating cancer, the methodincluding a step of administering a therapeutically effective amount ofthe recombinant adenovirus to an individual.

Since the pharmaceutical composition of the present invention uses theabove-described recombinant adenovirus, the description of the contentcommon between the two will be omitted in order to avoid the excessivecomplexity of the present specification.

As used herein the term “prevention” refers to all actions that suppresscancer (tumors) or delay the onset of the cancer (tumors) byadministering the pharmaceutical composition according to the presentinvention.

As used herein, the term “treatment” refers to all actions thatameliorate or beneficially change symptoms for cancer (tumors) byadministering the pharmaceutical composition according to the presentinvention.

In the present invention, “an individual” refers to a subject in need oftreatment of a disease, and more specifically, refers to a mammal suchas a human or a non-human primate, a rodent (a rat, a mouse, a guineapig, and the like), a mouse, a dog, a cat, a horse, a cow, a sheep, apig, a goat, a camel, and an antelope.

“Cancer”, which is a disease to be prevented or treated by thepharmaceutical composition of the present invention, collectively refersto diseases caused by cells having aggressive characteristics in whichthe cells ignore normal growth limits and divide and grow, invasivecharacteristics to infiltrate surrounding tissues, and metastaticcharacteristics of spreading to other sites in the body. In the presentinvention, the cancer is used in the same sense as a malignant tumor,and may comprise a solid tumor and a blood borne tumor. For example, thecancer may be any one selected from the group consisting of gastriccancer, lung cancer, non-small cell lung cancer, breast cancer, ovariancancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngealcancer, pancreatic cancer, bladder cancer, colon cancer, cervicalcancer, bone cancer, non-small cell bone cancer, hematologic malignancy,skin cancer, head or neck cancer, uterine cancer, colorectal cancer,anal near cancer, colon cancer, fallopian tube cancer, endometrialcancer, vaginal cancer, vulva cancer, Hodgkin's disease, esophagealcancer, small intestine cancer, endocrine cancer, thyroid cancer,parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethralcancer, penile cancer, prostate cancer, chronic or acute leukemia,lymphocytic lymphoma, kidney or hydroureter cancer, renal cellcarcinoma, renal pelvic carcinoma, salivary gland cancer, sarcomacancer, pseudomyxoma peritonei, hepatoblastoma, testicular cancer,glioblastoma, cheilocarcinoma, ovarian germ cell tumors, basal cellcarcinoma, multiple myeloma, gallbladder cancer, choroidal melanoma,cancer of the ampulla of Vater, peritoneal cancer, tongue cancer, smallcell cancer, pediatric lymphoma, neuroblastoma, duodenal cancer,ureteral cancer, astrocytoma, meningioma, renal pelvis cancer, pudendumcancer, thymus cancer, central nervous system (CNS) tumors, primarycentral nervous system lymphoma, spinal cord tumors, brain stemneuroglioma, and pituitary adenoma, and may also be a recurrent cancer,but is not limited thereto.

Meanwhile, the pharmaceutical composition of the present invention mayadditionally comprise an immune checkpoint inhibitor.

As used herein, the term “immune checkpoint” collectively refers to aprotein involved in causing stimulating or suppressing signals of animmune response on the surface of immune cells, and cancer cells evadethe surveillance network of the immune system by being manipulated suchthat the stimulation of the immune response and the resulting inhibitionof cancer cells are not properly performed through the immunecheckpoint. Preferably, the immune checkpoint protein may be a PD-1antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CD27 antagonist, aCD28 antagonist, a CD70 antagonist, a CD80 antagonist, a CD86antagonist, a CD137 antagonist, a CD276 antagonist, a KIRs antagonist, aLAG3 antagonist, a TNFRSF4 antagonist, a GITR antagonist, a GITRLantagonist, a 4-1BBL antagonist, a CTLA-4 antagonist, an A2ARantagonist, a VTCN1 antagonist, a BTLA antagonist, an IDO antagonist, aTIM-3 antagonist, a VISTA antagonist, a KLRA antagonist, and acombination thereof, but is not limited thereto.

The immune checkpoint inhibitor is an antagonist or antibody targetingthe immune checkpoint protein, and exhibits an anticancer effect causedby an immune response by enhancing a protein which stimulates the immuneresponse or blocking a protein which suppresses the immune response.Since the immune checkpoint inhibitor uses an immune response systemwhich is excellent in memory ability in addition to advantages of fewerside effects such as emesis or alopecia than general cytotoxicanticancer agents and large therapeutic effects, the therapeutic effectmay be sustained for a long period of time even after the administrationof a drug is stopped, but the enhancement of the anticancer effectthrough a co-administration with the recombinant adenovirus has not beenknown. Thus, the present inventors attempted co-administration with animmune checkpoint inhibitor in order to enhance the anticancer effect ofthe recombinant adenovirus, and there is another technical feature inthat there is provided a medicinal use of the recombinant adenovirus forthe prevention or treatment of cancer as a co-administration formulationwith the immune checkpoint inhibitor.

The pharmaceutical composition of the present invention may comprise apharmaceutically acceptable carrier in addition to the activeingredient. In this case, the pharmaceutically acceptable carrier istypically used during the formulation, and comprises lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate,alginate, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidinone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,mineral oil, and the like, but is not limited thereto. Furthermore, thepharmaceutically acceptable carrier may further comprise a lubricant, awetting agent, a sweetening agent, a flavoring agent, an emulsifier, asuspending agent, a preservative, and the like, in addition to theaforementioned ingredients. Suitable pharmaceutically acceptablecarriers and formulations are described in detail in Remington'sPharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may beadministered orally or parenterally (for example, intravenously,subcutaneously, intraperitoneally, or applied topically), and accordingto an example of the present invention, the anti-tumor immunityenhancing composition of the present invention may be preferablydirectly administered intratumorally. The dose varies depending on thepatient's condition and body weight, severity of the disease, drug form,administration route, and duration, but may be suitably selected bythose skilled in the art.

The pharmaceutical composition of the present invention is administeredin a pharmaceutically effective amount. In the present invention,“pharmaceutically effective amount” means an amount sufficient to treatdiseases at a reasonable benefit/risk ratio applicable to medicaltreatment, and an effective dosage level may be determined according tofactors including type of disease of patients, the severity of disease,the activity of drugs, sensitivity to drugs, administration time,administration route, excretion rate, treatment period, andsimultaneously used drugs, and other factors well known in the medicalfield. Another pharmaceutical composition according to the presentinvention may be administered as an individual therapeutic agent or incombination with other therapeutic agents, may be administeredsequentially or simultaneously with therapeutic agents in the relatedart, and may be administered in a single dose or multiple doses. It isimportant to administer the composition in a minimum amount that canobtain the maximum effect without any side effects, in consideration ofall the aforementioned factors, and this amount may be easily determinedby those skilled in the art.

Hereinafter, preferred Examples for helping the understanding of thepresent invention will be suggested. However, the following Examples areprovided only to more easily understand the present invention, and thecontents of the present invention are not limited by the followingExamples.

EXAMPLES Experimental Materials and Experimental Methods

1. Animal Experiment

6-week to 8-week old male C57BL/6 mice were purchased from Charles RiverLaboratories International, Inc. (Wilmington, Mass.) and bred in alaminar air flow cabinet under a pathogen free condition. All animalexperiments were carried out under the approval of the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC), andperformed in accordance with the guidelines established by theUniversity of Hanyang Institutional Animal Care and Use Committee.

2. Manufacture of Oncolytic Adenovirus

An adenovirus (Ad), in which IL-12 and GM-CSF-Relaxin or shVEGF wereintroduced into the E1 region and the E3 region, respectively, wasmanufactured as illustrated in FIGS. 1 and 2.

Specifically, in order to construct an Ad E1 shuttle vector expressingIL-12, a pXC1RdB/IL12 E1 shuttle vector was manufactured by excising amouse IL-12 gene from pCA14/IL12 and sub-cloning the gene into a pXC1RdBE1 shuttle vector. Further, in order to construct a shVEGF-expressing E3shuttle vector, full-length mouse shVEGF complementary DNA was cloned byRT-PCR using total RNA obtained from bone marrow-derived activedendritic cells. The shVEGF complementary DNA (nucleotides 53-982 of theNational Center for Biotechnology Information L15435) was manufacturedusing the following primer set: sense(5′-gatcccggaaggagagcagaagtcccatgttcaagagacatgggacttctgctctcctttttttttggaaa-3′),antisense (5′-tttccaaaaaaaaaggagagcagaagtcccatgtctcttgaacatgggacttctgctctccttccgggatc-3′). The PCRproduct was digested with BamHI/Hind III, and then cloned in aBamHI/Hind III-treated pSP72 E3/CMV-polA Ad E3 shuttle vector, therebymanufacturing a pSP72 E3/shVEGF E3 shuttle vector. Further, a pSP72E3/GMCSF-RLX E3 shuttle vector was manufactured by cloningGMCSF-IRES-Relaxin gene in a pSP72 E3/CMV-polA Ad E3 shuttle vector.

For homologous recombination, E. coli BJ5183 was transformedsimultaneously with the pXC1RdB/IL12 E1 shuttle vector and pdE1/shVEGFor pdE1/GMCSF-RLX, thereby manufacturing pRdB/IL12/shVEGF Ad andpRdB/IL12/GMCSF-RLX Ad. All viruses were manufactured using 293 cells,and the purification, titration, and quality analysis of the adenoviruswere carried out according to the prior art.

3. Evaluation of Anticancer Effect Using Animal Model

B16-F10 cells (5×10⁵) were injected subcutaneously into the right flankof 6- to 7-week old male C57BL/6 immune-competent mice. When the tumorvolume reached approximately 100 mm³, the mice were divided into groupswith similar tumor sizes, and the oncolytic adenoviruses(RdB/IL12/shVEGF Ad; RdB/IL12/GMCSF-RLX Ad) were administeredintratumorally at a concentration of 5×10⁹ VP on day 1, 3, and 5. Inaddition, in order to confirm the anticancer effect according to theco-administration with an immune checkpoint inhibitor, an anti PD-L1antibody, an anti PD-1 antibody, and an anti CTLA-4 antibody as immunecheckpoint inhibitors were administered intraperitoneally at aconcentration of 200 μg to the oncolytic adenovirus-administered mice onday 3, 6, and 9, and in order to further evaluate a synergistic effectby the co-administration with the immune checkpoint inhibitor, the sameexperiment as described above was performed again using the oncolyticadenovirus at a dose (1×10⁹ VP) which is five times lower than that ofthe previous experiment using 5×10⁹ VP. Meanwhile, in the presentexperiment, a PBS-treated group (PBS) and groups treated with the immunecheckpoint inhibitor alone (anti PD-L1 antibody, anti PD-1 antibody, andanti CTLA-4 antibody) were used as controls.

Thereafter, an anticancer effect was evaluated using an animal model bymeasuring a perpendicular tumor diameter using calipers to monitor thegrowth of tumors every day and confirming a change in survival rate overtime. Meanwhile, tumor volume was calculated using the followingformula: volume=0.523 L (W)², in which L indicates a length and Windicates a width.

4. Evaluation of Anticancer Effect by Immune Memory

On day 50 after a primary tumor was injected into 3 animal models inwhich melanoma was induced, a secondary tumor was reinjected(rechallenge) into successfully treated mice (on day 25 from the timepoint when the primary tumor could not be promoted). Thereafter, in thesame manner as described above, an anti-tumor immune memory effect wasevaluated by measuring a perpendicular tumor diameter to monitor thegrowth of tumors every other day and calculating an average tumorvolume. In the present experiment, a group in which melanoma was inducedin normal mice (Normal) was used as a control.

5. Statistical Analysis

All data was expressed as mean±standard error. Comparisons were madeusing Stat View software (Abacus Concepts, Inc., Berkeley, Calif.) andthe Mann-Whitney test (non-parametric rank sum test). P values equal toor less than 0.05 indicate statistically significance (*, P<0.05; **,P<0.01).

Experimental Results

1. Confirmation of Anticancer Effect in Melanoma Mice

The present inventors intended to confirm the anticancer effect of therecombinant adenovirus and/or the immune checkpoint inhibitor bycomparing the volume of a tumor injected into melanoma mice, thepresence of complete remission, and the survival rate of the mice.

As a result, it was confirmed that in the control treated with PBS orthe anti PD-L1 antibody, tumors had proliferated rapidly enough for thetumor volume to reach 3,000 mm³ or more on day 11 or 15, respectively,thereby exhibiting aggressive tumor growth, whereas in the group treatedwith RdB/IL12/shVEGF Ad or RdB/IL12/GMCSF-RLX Ad, as a result ofcomparison with the PBS-treated group on day 22, tumor growth wassuppressed by 99.3% or 99.5% (FIGS. 3A and 4A), respectively.Furthermore, when the adenovirus and the anti PD-L1 antibody wereco-administered (RdB/IL12/shVEGF Ad+PD-L1, RdB/IL12/GMCSF-RLX Ad+PD-L1),the tumor volume was more remarkably decreased than that of the grouptreated with the adenovirus or the anti PD-L1 antibody alone, in thesame manner as described above. Although an anticancer effect similar tothat of the group treated with the adenovirus alone (RdB/IL12/shVEGF Ad,RdB/IL12/GMCSF-RLX Ad) was exhibited until day 27, from the time whenone month had passed, the growth of tumors was continuously suppressedin the group to which the anti PD-L1 antibody was co-administered,whereas in the group treated with the adenovirus alone, the growth oftumors was increased over time, so that a significant difference intumor volume could be confirmed between the group treated with theadenovirus alone and the group to the adenovirus and the antibody wereco-administered (FIGS. 3A and 4A). In addition, it could be seen that onday 50, in the control treated with PBS or the anti PD-L1 antibody,complete remission could not be confirmed, whereas in theRdB/IL12/shVEGF Ad or RdB/IL12/GMCSF-RLX Ad group, the survival rateswere 66% (4/6) and 50% (3/6), respectively, and when the adenovirus andthe anti PD-L1 antibody were co-administered, the survival rates were66% (4/6) and 100% (6/6) which is complete remission. Furthermore, itwas confirmed that all the mice in the RdB/IL12/shVEGF Ad+PD-L1 orRdB/IL12/GMCSF-RLX Ad+PD-L1 group survived until day 44 or day 50,respectively, whereas at the same time point, the RdB/IL12/shVEGF Ad orRdB/IL12/GMCSF-RLX Ad group exhibited a survival rate of 66% (4/6), andall the mice in the control were killed by tumors (FIGS. 3B and 4B).These results mean that the anticancer effect by treatment with theimmune checkpoint inhibitor anti PD-L1 antibody alone was relativelyinsignificant, whereas RdB/IL12/shVEGF Ad or RdB/IL12/GMCSF-RLX Adexhibited an excellent anticancer effect, and the anticancer effect ofthe recombinant adenovirus is further enhanced when co-administered withthe anti PD-L1 antibody.

2. Anticancer Effect by Co-Administration with Immune CheckpointInhibitor

The present inventors compared the anticancer effects between onlyadministration of the recombinant adenovirus and the co-administrationof the recombinant adenovirus and the immune checkpoint inhibitor usingthe oncolytic adenovirus at a dose (1×10⁹ VP) which is five times lowerthan that of the previous experiment using 5×10⁹ VP, in order toevaluate the synergistic effect by the co-administration with the immunecheckpoint inhibitor in more detail. Further, based on the results, whenthe anti PD-L1 antibody, the anti PD-1 antibody, or the anti CTLA-4antibody was used as the immune checkpoint inhibitor, the anticancereffects of these antibodies were compared, and in the presentexperiment, RdB/IL12/GMCSF-RLX Ad was used as the recombinantadenovirus.

As a result, on day 18, as a result of comparison with the tumor volumeof the PBS-treated group, the tumor growth suppression rate of theRdB/IL12/shVEGF Ad group was 92.9%, but the tumor growth suppressionrate was increased to 99.7% by the co-administration with the anti PD-1antibody, and the tumor growth suppression rate of theRdB/IL12/GMCSF-RLX Ad group (90.1%) was also greatly increased to 98.9%by the co-administration with the anti PD-1 antibody (FIGS. 5 and 6). Inaddition, only 17% (1/6) of the RdB/IL12/shVEGF Ad group underwentcomplete remission, but in the RdB/IL12/shVEGF Ad+PD-1 group, thecomplete remission rate was 83% (5/6), which is greatly increased.

Meanwhile, in order to confirm whether other types of recombinantadenoviruses also exhibited the synergistic effect by theco-administration with the immune checkpoint inhibitor,HmT-Rd19-K35/IL21 Ad, RdB/GMCSF/shVEGF Ad, or YKL-1/GMCSF/B7.1 Ad andthe anti PD-1 antibody were co-administered, and then the anticancereffects thereof were confirmed. As a result, as illustrated in FIG. 7,the fact was that in most of the recombinant adenoviruses, the tumorvolume was decreased to some degree through the co-administration of theanti PD-1 antibody, but most of the recombinant adenoviruses failed toexhibit a remarkable effect as much as the recombinant adenovirus of thepresent invention (RdB/IL12/shVEGF Ad, RdB/IL12/GMCSF-RLX Ad) did, andin particular, the effect of YKL-1/GMCSF/B7.1 Ad on the whole was notexcellent as compared to that of the group treated with the anti PD-1antibody alone. From these results, it can be seen that the synergisticeffect by the co-administration with the immune checkpoint inhibitor isnot applicable to all recombinant adenoviruses, and is a unique effectof the recombinant adenovirus of the present invention.

Further, it was confirmed that as a result of confirming the anticancereffect by the type of immune checkpoint inhibitor, the control micetreated with PBS, the anti PD-L1 antibody, the anti PD-1 antibody, orthe anti CTLA-4 antibody had proliferated rapidly enough for the tumorvolume to reach 3,000 mm³ or more on day 11, 15, 18, or 11,respectively, thereby exhibiting aggressive tumor growth, whereas in thecase of the groups treated with RdB/IL12/GMCSF-RLX Ad+anti PD-L1antibody, RdB/IL12/GMCSF-RLX Ad+anti PD-1 antibody, andRdB/IL12/GMCSF-RLX Ad+anti CTLA-4 antibody, on day 21, as a result ofcomparison with the PBS-treated group, tumor growth was greatlysuppressed by 99.9%, 99.9%, or 99.2%, respectively (FIGS. 4A, 8A, and9A). In addition, on day 50, in the RdB/IL12/GMCSF-RLX Ad group, thecomplete remission rate was 50% (3/6), whereas when the anti PD-L1antibody, the anti PD-1 antibody, or the anti CTLA-4 antibody wasco-administered, the complete remission rate was 100% (6/6), 83% (5/6),and 83% (5/6), respectively, which are increased. Furthermore, micetreated with RdB/IL12/GMCSF-RLX Ad+anti PD-L1 antibody,RdB/IL12/GMCSF-RLX Ad+anti PD-1 antibody, and RdB/IL12/GMCSF-RLX Ad+antiCTLA-4 antibody survived until day 50, day 50, or day 30, respectively,whereas at the same time points, the RdB/IL12/GMCSF-RLX Ad groupexhibited a survival rate of 66% (4/6), 66 (4/6), and 83% (5/6),respectively (FIGS. 4B, 8B, and 9B). These results show that even whennot only the anti PD-L1 antibody, but also the anti PD-1 antibody, orthe anti CTLA-4 antibody are used, the anticancer effect of therecombinant adenovirus is enhanced, however, it could be seen that whenthe anti PD-L1 antibody or the anti PD-L1 antibody was co-administered,the anticancer effect-associated synergistic effect ofRdB/IL12/GMCSF-RLX Ad was more predominant than that when the antiCTLA-4 antibody was co-administered.

3. Confirmation of Immune Memory Effect Against Recurrent Cancer

The present inventors intended to confirm an immune memory effect and ananticancer effect against a recurrent cancer according to therecombinant adenovirus of the present invention, or theco-administration of the recombinant adenovirus and the immunecheckpoint inhibitor by comparing the volume of secondary tumorsinjected into melanoma mice, and the like.

As a result, it could be confirmed that in the RdB/IL12/shVEGF Ad group,the growth of secondary tumors was markedly inhibited (FIG. 10A), andparticularly, the growth of secondary tumors was more remarkablyinhibited by the co-administration with the immune checkpoint inhibitoranti PD-1 antibody (FIG. 10B). These results mean that the recombinantadenovirus of the present invention may prevent the recurrence of tumorsin advance through the effect of enhancing the anti-tumor immuneresponse by immune memory, suggesting that the effect may be enhanced bythe co-administration with the immune checkpoint inhibitor as with theabove-described anticancer effect.

4. Verification of Anticancer Effect of RdB/IL12/GMCSF-RLX Ad

(1) Confirmation of Expression Patterns of IL-12, GM-CSF, and Relaxin

In order to verify the anticancer effect according to the co-expressionof IL-12, GM-CSF, and relaxin (RLX) genes, RdB/IL12/GMCSF-RLX Ad, whichis an adenovirus in which the IL-12 gene was inserted into the E1 regionof an adenovirus and GM-CSF and relaxin genes were inserted into the E3region of the adenovirus, was manufactured as described above. TheRdB/IL12/GMCSF-RLX Ad is a cancer cell-specific oncolytic adenovirus inwhich an E1B gene as an initial gene of an adenovirus was deleted and anE1A gene was modified, and the expression level of IL-12, GM-CSF, or RLXby the manufactured adenovirus was confirmed. Specifically, a Syrianhamster pancreatic cancer cell line (Hap-T1) was infected withRdB/IL12/GMCSF-RLX at a multiplicity of infection (MOI) of 0.2, 0.5, 1,2, and 5, and 48 hours later, the media thereof were collected.Thereafter, the expression levels of IL-12 and GM-CSF were confirmedthrough enzyme-linked immunosorbent assay (ELISA), and the expressionlevel of relaxin was confirmed through a reverse transcriptionpolymerase chain reaction (RT-PCR).

As a result, as illustrated in FIGS. 11 and 12, the expression level ofIL-12, GM-CSF, or relaxin was increased dependently on the MOI of theadenovirus, so that the co-expression of the genes could beexperimentally confirmed.

(2) Enhancement of anticancer effect by co-administration with αPD-1 inSyrian hamster tumor animal model

In order to verify the enhancement of the anticancer effect by theco-administration of RdB/IL12/GMCSF-RLX Ad and αPD-1, 7×10⁷ viralparticles (VP)/30 μl of RdB/IL12/GMCSF-RLX Ad was administeredintratumorally to a Syrian hamster tumor animal model induced bysubcutaneously injecting a HaP-T1 pancreatic cancer cell line, and alongwith this, 10 mg/kg of αPD-1 was administered intraperitoneally, andthen the resulting change in volume of tumors was observed. Meanwhile,an RdB/IL12/GMCSF-RLX Ad only administration group was used as acomparative group.

As a result, as illustrated in FIG. 13, in the tumor tissue to whichRdB/IL12/GMCSF-RLX Ad was administered alone, tumors grew continuously,so that on day 30 after the first administration of the virus, the tumorvolume reached 1.982±126 mm³, whereas in the co-administration group ofRdB/IL12/GMCSF-RLX Ad and αPD-1, the growth of tumors was suppresseduntil day 30 after the first administration of the virus, therebysuppressing the growth of tumors to a level of about 79% as compared tothe single administration group. Further, in the co-administration groupof RdB/IL12/GMCSF-RLX Ad and αPD-1, the complete remission rate oftumors was about 50%, but in the RdB/IL12/GMCSF-RLX Ad onlyadministration group, the complete remission was not observed at all(not illustrated). That is, these results suggest that the anticancertherapeutic efficacy of RdB/IL12/GMCSF-RLX Ad is remarkably improved bythe co-administration with αPD-1.

(3) Change in Tumor Tissue by Co-Administration with αPD-1 in SyrianHamster Tumor Animal Model

In order to specifically confirm the change in tumor tissues by theco-administration of RdB/IL12/GMCSF-RLX Ad and αPD-1, after 7×10⁷ VP ofRdB/IL12/GMCSF-RLX Ad and 10 mg/kg of αPD-1 were co-administered asdescribed above, the collected tumor tissues were immunohistologicallyevaluated. Meanwhile, an αPD-1 or RdB/IL12/GMCSF-RLX Ad onlyadministration group and a PBS administration group were used as acomparative group and a control, respectively.

As a result, as illustrated in FIG. 14, it could be confirmed that inthe tumor tissue to which PBS was administered, a necrosis site washardly confirmed, whereas most of the tumor tissues to whichRdB/IL12/GMCSF-RLX Ad and αPD-1 were co-administered became necrotic.Further, the effect by the co-administration, that is, the remarkablylow proliferation of tumor cells (PCNA) and the increased cell necrosis(TUNEL) were very remarkable as compared to the case where αPD-1 orRdB/IL12/GMCSF-RLX Ad was administered alone.

(4) Change in Expression of Cell Populations and IFN-γ in Tumor Tissuesby Co-Administration with αPD-1

In order to evaluate whether the infiltration and activation of T cellsin tumor tissues could be induced as the infiltration of αPD-1 in tumortissues was increased by RdB/IL12/GMCSF-RLX Ad, a change in populationof interferon (IFN)-y-expressing CD4⁺ or CD8⁺ T cells was evaluated inthe infiltrated lymphocytes in the draining lymph node (DLN) and tumortissues. As a result, as illustrated in FIGS. 15 and 16, in theRdB/IL12/GMCSF-RLX Ad or αPD-1 only administration group and theco-administration group of RdB/IL12/GMCSF-RLX Ad and αPD-1, thepopulation of IFN-γ-expressing CD4⁺ and CD8⁺ T cells was observed at ahigh level as compared to the PBS or αPD-1 only administration group(P<0.001 or P<0.01). In particular, it could be confirmed that in theco-administration group of RdB/IL12/GMCSF-RLX Ad and αPD-1, thepopulation of IFN-γ-expressing CD4⁺ and CD8⁺ T cells was alsosignificantly increased as compared to the group to whichRdB/IL12/GMCSF-RLX Ad or αPD-1 was administered alone (P<0.001), andthis result indicates that the co-administration of RdB/IL12/GMCSF-RLXAd and αPD-1 induces the infiltration and activation of interferon(IFN)-γ-expressing CD4⁺ or CD8⁺ T cells in tumors.

In addition, in order to evaluate the expression level of IFN-γ in thetumor tissue to which the adenovirus was administered, tumor tissueswere extracted from mice to which the adenovirus and the like wereadministered and finely ground, and then an IFN-γ ELISA analysis wascarried out. As a result, as illustrated in FIG. 17, it could beconfirmed that when PBS or αPD-1 was administered intratumorally alone,IFN-γ was not detected at all, and in the RdB/IL12/GMCSF-RLX Ad onlyadministration group, only 8.3 pg/mg of IFN-γ per 1 g of the tumortissue was detected, whereas in the co-administration group ofRdB/IL12/GMCSF-RLX Ad and αPD-1, 18.7 pg/mg of IFN-γ per 1 g of thetumor tissue was detected. That is, it was revealed that theaforementioned co-administration increased the activity of immune cellsby increasing the expression of IFN-γ in tumors, and ultimately,contributed to the enhancement of the anticancer effect.

(5) Confirmation of Anticancer Effect Under Condition of Resistance toImmune Checkpoint Inhibitor

In order to evaluate whether the co-administration of RdB/IL12/GMCSF-RLXAd and αPD-1 could exhibit effective therapeutic efficacy under acondition of resistance to the immune checkpoint inhibitor, the effectof the co-administration to tumors pre-treated with αPD-1 on suppressionof the growth of tumors was evaluated. Specifically, when the growth oftumors was not effectively suppressed by the administration of αPD-1alone (on day 9 after the first treatment of the antibody),RdB/IL12/GMCSF-RLX Ad was administered four times in total at aninterval of 2 days. Meanwhile, an αPD-1 only administration group wasused as a comparative group.

As a result, as illustrated in FIG. 18, the administration ofRdB/IL12/GMCSF-RLX Ad initiated when the tumor volume reached 915±78 mm³effectively suppressed the growth of tumors for 10 days from the firstday of the administration. Interestingly, the αPD-1 only administrationgroup exhibited a rapid increase in tumor growth rate, and thus failedto suppress the growth of tumors, whereas the co-administration groupcontinuously suppressed the growth of tumors even during theaforementioned period. Further, as a result of comparing a change intumor volume (on day 23 after the administration), the effect ofsuppressing the growth in the co-administration group was the mostremarkable. That is, the aforementioned co-administration exhibitedeffective anticancer therapeutic efficacy even under a condition ofresistance to the immune checkpoint inhibitor. Accordingly, it issuggested that the aforementioned co-administration may become a therapybeneficial to a patient having tolerance to an immune checkpointinhibitor single therapy.

(6) Change in Infiltration of αPD-1 into Tumor Tissues by Administrationof RdB/IL12/GMCSF-RLX Ad

It was predicted that the expression of RLX in tumor tissues wouldexhibit synergistic anticancer therapeutic efficacy by promoting theintratumoral infiltration of αPD-1. Accordingly, it was intended toevaluate whether RdB/IL12/GMCSF-RLX Ad increased the intratumoralinfiltration of αPD-1 into a desmoplastic pancreatic tumor tissue.RdB/IL12/GMCSF-RLX Ad was directly administered to tumor tissues on day0 or day 2, αPD-1 was administered intraperitoneally on day 2 (c:RdB/IL12/GMCSF-RLX+Alexa 488-anti PD-1), and an Alexa 488-conjugatedαPD-1 only administration group (b; Alexa 488-anti PD-1) and a PBS onlyadministration group (a; PBS) were used as a comparative group and acontrol, respectively. The tumor tissue treated as described above wascollected on day 7, and the accumulation of Alexa 488-αPD-1 in the tumortissue was quantified through an immunofluorescence analysis. As aresult of quantifying the accumulation of Alexa 488-αPD-1 per tissuearea, as illustrated in FIG. 20, the PBS only administration group orαPD-1 only administration group exhibited 14±2 A.U./μm² or 21±1A.U./μm², respectively, whereas the co-administration group exhibited32±6 A.U./μm². From the result, it could be seen that RdB/IL12/GMCSF-RLXAd remarkably improved the infiltration of αPD-1 into tumor tissues andcontributed to the improvement of localization of αPD-1 in tumortissues. Furthermore, in order to confirm whether the Alexa 488-αPD-1was located at the expression site of CD4⁺ or CD8⁺ T cells, with respectto each tumor tissue to which PBS (a: PBS) or Alexa 488-conjugated αPD-1(b: Alexa 488-anti PD-1) are administered alone or RdB/IL12/GMCSF-RLX Adand Alexa 488-conjugated αPD-1 were co-administered (c:RdB/IL12/GMCSF-RLX+Alexa 488-anti PD-1), immunostaining was carried outon CD4+CD8+. As a result, as illustrated in FIG. 21, in theco-administration group, the co-localization of αPD-1 and CD4⁺ or CD8⁺ Tcells were observed at a higher level than in the Alexa 488-αPD-1 onlyadministration group.

In order to evaluate the intratumoral infiltration of αPD-1 for thewhole body, ⁶⁴Cu-αPD-1 in which αPD-1 and ⁶⁴Cu were conjugated wasadministered along with RdB/IL12/GMCSF-RLX Ad to HaP-Ti-transplantedhamsters using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidby the above-described method, thereby obtaining an immuno-PET image.PET scanning was carried out at 2, 12, 36, and 60 hours after theadministration of ⁶⁴Cu-αPD-1, and main PET imaging was carried out alongwith CT for anatomical description. A ⁶⁴Cu-αPD-1 only administrationgroup was used as a comparative group. As a result, as illustrated inFIG. 22, the intratumoral absorption of ⁶⁴Cu-αPD-1 by theco-administration group and the single administration group calculatedas an SUV was as follows: 0.26±0.06 and 0.36±0.05 (P=0.072) at 2 hours;0.90±0.22 and 1.68±0.45 (P=0.018) at 12 hours; 2.14±0.19 and 2.97±0.67(P=0.062) at 36 hours; and 3.37±0.57 and 4.50±1.02 (P=0.043) at 60hours. That is, it was observed that at all the aforementioned timepoints, the intratumoral absorption of ⁶⁴Cu-αPD-1 by theco-administration group was significantly higher than that by the⁶⁴Cu-αPD-1 only administration group. Further, the distribution orabsorption of ⁶⁴Cu-αPD-1 in various tissues including tumor tissues wascalculated as % ID/g at 60 hours after the administration of ⁶⁴Cu-αPD-1.As a result, as illustrated in FIG. 23, it could be confirmed that theabsorption exhibited by the single administration group and theco-administration group was: 1.80±0.13% ID/g, 2.80±0.41% ID/g (P<0.05),respectively in tumor tissues; and 1.10±0.11% ID/g, 1.94±0.23% ID/g(P<0.05), respectively in the spleen, and in the co-administrationgroup, the absorption of ⁶⁴Cu-αPD-1 was improved in both tumor tissuesand the spleen. Furthermore, in addition to these tissues, the draininglymph node (DLN) known as a T cell-enriched site was enlarged byinfection with RdB/IL12/GMCSF-RLX Ad (result not illustrated). That is,when the aforementioned experimental results are taken together, itcould be seen that the infection with RdB/IL12/GMCSF-RLX Ad remodeled ordecomposed the extracellular matrix in tumor tissues and improved theabsorption of αPD-1 in tumor tissues by the enhanced immune response byIL-12 and GM-CSF, and as a result, a synergistic effect of theco-administration occurred.

The above-described description of the present invention is provided forillustrative purposes, and the person skilled in the art to which thepresent invention pertains will understand that the present inventioncan be easily modified into other specific forms without changing thetechnical spirit or essential features of the present invention.Therefore, it should be understood that the above-described Examples areillustrative only in all aspects and are not restrictive.

What is claimed is:
 1. A recombinant oncolytic adenovirus comprising: agene encoding Interleukin 12 (IL-12); and a gene encoding a shRNAsuppressing the expression of VEGF (Vascular endothelial growth factor).2. The recombinant oncolytic adenovirus of claim 1, wherein therecombinant adenovirus has any one or more regions selected from thegroup consisting of E1B and E3 regions deleted.
 3. The recombinantoncolytic adenovirus of claim 2, wherein in the recombinant adenovirusthe gene encoding IL-12 is inserted into the E1B region, and the geneencoding a shRNA suppressing the expression of VEGF (Vascularendothelial growth factor) is inserted into the E3 region.
 4. Apharmaceutical composition for preventing or treating cancer, thecomposition comprising: (a) the recombinant oncolytic adenovirus ofclaim 1; and (b) a pharmaceutically acceptable carrier.
 5. Thepharmaceutical composition of claim 4, wherein the composition furthercomprises immune checkpoint inhibitors.
 6. The pharmaceuticalcomposition of claim 5, wherein the immune checkpoint inhibitor is anyone selected from the group consisting of a programmed cell death-1(PD-1) antagonist, a programmed cell death-ligand 1 (PD-L1) antagonist,a programmed cell death-ligand 2 (PD-L2) antagonist, a cluster ofdifferentiation 27 (CD27) antagonist, a cluster of differentiation 28(CD28) antagonist, a cluster of differentiation 70 (CD70) antagonist, acluster of differentiation 80 (CD80, also known as B7-1) antagonist, acluster of differentiation 86 (CD86, also known as B7-2) antagonist, acluster of differentiation 137 (CD137) antagonist, a cluster ofdifferentiation 276 (CD276) antagonist, a killer-cellimmunoglobulin-like receptors (KIRs) antagonist, a lymphocyte-activationgene 3 (LAG3) antagonist, a tumor necrosis factor receptor superfamily,member 4 (TNFRSF4, also known as CD134) antagonist, aglucocorticoid-induced TNFR-related protein (GITR) antagonist, aglucocorticoid-induced TNFR-related protein ligand (GITRL) antagonist, a4-1BB ligand (4-1BBL) antagonist, a cytolytic T lymphocyte associatedantigen-4 (CTLA-4) antagonist, an adenosine A2A receptor (A2AR)antagonist, a V-set domain-containing T-cell activation inhibitor 1(VTCN1) antagonist, a B- and T-lymphocyte attenuator (BTLA) antagonist,an indoleamine 2,3-dioxygenase (IDO) antagonist, a T-cell Immunoglobulindomain and Mucin domain 3 (TIM-3) antagonist, a V-domain Ig suppressorof T cell activation (VISTA) antagonist, a killer cell lectin-likereceptor subfamily A (KLRA) antagonist, and a combination thereof. 7.The pharmaceutical composition of claim 4, wherein the cancer is any oneselected from the group consisting of gastric cancer, lung cancer,non-small cell lung cancer, breast cancer, ovarian cancer, liver cancer,bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreaticcancer, bladder cancer, colon cancer, cervical cancer, bone cancer,non-small cell bone cancer, hematologic malignancy, skin cancer, headand neck cancer, uterine cancer, colorectal cancer, anal near cancer,fallopian tube cancer, endometrial cancer, vaginal cancer, vulva cancer,Hodgkin's disease, esophageal cancer, small intestine cancer, endocrinecancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissuesarcoma, urethral cancer, penile cancer, prostate cancer, chronic oracute leukemia, lymphocytic lymphoma, kidney or hydroureter cancer,renal cell carcinoma, renal pelvic carcinoma, salivary gland cancer,sarcoma cancer, pseudomyxoma peritonei, hepatoblastoma, testicularcancer, glioblastoma, cheilocarcinoma, ovarian germ cell tumors, basalcell carcinoma, multiple myeloma, gallbladder cancer, choroidalmelanoma, cancer of the ampulla of Vater, peritoneal cancer, tonguecancer, small cell cancer, pediatric lymphoma, neuroblastoma, duodenalcancer, astrocytoma, meningioma, renal pelvis cancer, pudendum cancer,thymus cancer, central nervous system (CNS) tumors, primary centralnervous system lymphoma, spinal cord tumors, brain stem neuroglioma, andpituitary adenoma.
 8. The pharmaceutical composition of claim 4, whereinthe cancer is a recurrent cancer.
 9. The pharmaceutical composition ofclaim 4, wherein the composition enhances anti-tumor immunity.
 10. Amethod for treating cancer comprising administering a compositioncomprising the recombinant oncolytic adenovirus of claim 1; and apharmaceutically acceptable carrier, to an individual.
 11. The methodaccording to claim 10, wherein the composition is administratedsimultaneously, separately, or sequentially in combination with animmune checkpoint inhibitor.